Method of preventing acute or sub-acute hepatic failure by administering a long-acting recombinant human soluble tumor necrosis factor alpha receptor II fusion protein

ABSTRACT

The present invention belongs to the field of the application of genetic engineering and gene function, and it is directed to a new medical use of the gene encoding the recombinant soluble tumor necrosis factor α receptor (HusTNFR). The present invention made intervention to fulminant hepatic failure in mice by use of the long-acting recombinant human soluble tumor necrosis factor αreceptor and the classic animal models of acute and sub-acute hepatic failure. The results showed that the long-acting soluble tumor necrosis factor αreceptor of the present invention has a half-life extended more than 10 times, and it significantly decreased the mortality of model animals and has superior therapeutic effect for the treatment and/or prophylaxis of acute and sub-acute hepatic failure in model animals. These receptors have a noticeable therapeutic effect for the treatment and/or prophylaxis of acute and sub-acute hepatic failure in comparison with the non-long-acting HusTNFR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Division of application Ser. No. 12/090,037, filedApr. 11, 2008, which is a US National Stage Application of PCT/CN2006/002689, filed Oct. 13, 2006, which applications are incorporatedherein by reference. Application Ser. No. 12/090,037 is now U.S. Pat.No. 8,227,404.

TECHNICAL FIELD

The present invention belongs to the field of the application of geneticengineering and gene function. Particularly, it relates to a new medicaluse of the gene encoding the recombinant soluble tumor necrosis factorαreceptor (HusTNFR), and more particularly, it relates to use of thelong-acting recombinant human soluble tumor necrosis factor α receptor(LHusTNFR) in the treatment and/or prophylaxis of acute and sub-acutehepatic failure.

BACKGROUND ART

Fulminant hepatic failure (FHF) is a syndrome defined as necrosis of alarge number of liver cells or severe impairment of liver function andhepatic encephalopathy within 8 weeks after the initial symptom orwithin 10 days of the on-set of jaundice in the absence of previousliver disease. It is characterized by rapid on-set, severe condition,absence of effective treatment and high mortality.

The acute inflammation and necrosis of liver cells may lead to twodifferent diseases: acute hepatitis and fulminant hepatic failure (alsocalled acute hepatic failure). Acute hepatitis caused by hepatitis virusis referred to as acute viral hepatitis, and the acute hepatitis causedby alcohol is called as acute alcoholic hepatitis. Although necrosis ofliver cells is widely seen in all kinds of hepatitis described above,the necrosis is not so severe to lead to hepatic dysfunction. FHF isdistinguished from various acute hepatitis in that awfully rapidnecrosis of liver cells happens in FHF, resulting in hepatic failure dueto dysfunction of the remaining normal hepatocytes, and eventuallyleading to extremely high mortality.

The leading cause of FHF in China is hepatitis virus infection, whileother causes including medical or poison toxicosis, ischemia and anoxia,metabolic disorder, and autoimmune hepatitis, etc. Currently nomedicaments are available to specifically block the acute necrosis ofliver cells, and thus hepatic failure due to large quantities of hepaticcell necrosis cannot be prevented. As a result, it keeps ongoingdifficult to reduce the mortality of FHF, and there is an urgentclinical need for a medicament capable of specifically and rapidlypreventing acute hepatic cell necrosis, thereby treating hepaticfailure.

Recent studies have revealed that binding of TNFα and its receptor isthe first pathway to trigger hepatic cell necrosis, which plays animportant role in the mechanism of hepatocyte impairment. Blocking saidbinding will block the starting point of hepatic cell necrosis, wherebyenabling the pharmaceutical treatment of FHF.

At present, there are two classes of TNFαinhibitor that directlyinterrupt the binding of TNFαand its receptor: monoclonal antibodyagainst TNFα, and soluble TNFα receptor analogue. These TNFαinhibitors,which bind TNFαin vivo, will theoretically prevent the binding of TNFαin blood or intercellular fluid to its receptor on the membrane ofhepatocytes, thereby preventing the activation of the cell necrosispathway.

Recent studies found that monoclonal antibody of TNFα is not a suitablecandidate medicament due to its characteristic of activating TNFα oncell membrane which leads to apoptosis of the target cells, in additionto its ability to block the binding of TNFαto TNFα receptor type I.

The conventional soluble TNFαreceptors can to some extent decrease themortality in the most animal models of mild hepatic cell necrosis andacute hepatitis; however, it cannot effectively decrease the mortalityof acute or sub-acute hepatic failure caused by massive hepatocytenecrosis. Furthermore, the reason of the poor therapeutic effect isstill not known so far by those of the skill in the art. Consequently,it is still difficult now for the soluble TNFα receptors to find theiruse practically in clinical applications to treat massive hepatocytesnecrosis or hepatic failure.

In summary, there is an earnest demand in the art to find the reason forthe poor therapeutic effect of TNFαreceptors for treating hepatic cellnecrosis, so as to modify the TNFαreceptors to confer it the ability toeffectively and rapidly prevent the occurrence of extensive acutenecrosis of hepatocytes, and thereby making it a superior medicament forclinically treating and preventing acute and sub-acute hepatic failure.

Contents of the Invention

In the first aspect, the present invention provides a use of long-actingsoluble tumor necrosis factor αreceptor in the manufacture of amedicament for the treatment and/or prophylaxis of hepatocyte necrosisor hepatic failure.

In a preferred embodiment, the long-acting soluble tumor necrosis factorαreceptor is a long-acting recombinant human soluble tumor necrosisfactor αreceptor.

In a further preferred embodiment, the long-acting soluble tumornecrosis factor α receptor has a half-life of 12-140 hours (preferably,24-72 hours).

In a further preferred embodiment, the long-acting soluble tumornecrosis factor α receptor is selected from the group consisting of:

a. a fusion protein of human tumor necrosis factor αreceptor type I withhuman IgG1:Fc fragment (preferably, the carboxyl terminal of the humantumor necrosis factor αreceptor type I being linked to the aminoterminal of the IgG1:Fc fragment),

b. a fusion protein of human tumor necrosis factor αreceptor type IIwith human IgG1:Fc fragment (preferably, the carboxyl terminal of thehuman tumor necrosis factor α receptor type II being linked to the aminoterminal of the IgG1:Fc fragment),

c. a human tumor necrosis factor αreceptor type I protein PEGylated atthe amino terminal,

d. a human tumor necrosis factor αreceptor type I protein PEGylated atthe carboxyl terminal,

e. a human tumor necrosis factor αreceptor type II protein PEGylated atthe amino terminal,

f. a human tumor necrosis factor αreceptor type II protein PEGylated atthe carboxyl terminal,

h. a human tumor necrosis factor αreceptor type I protein embedded in aPEG-liposome mixture,

i. a human tumor necrosis factor α receptor type II protein embedded ina PEG-liposome mixture,

j. a fusion protein of human tumor necrosis factor αreceptor type I withhuman serum albumin, or

k. a fusion protein of human tumor necrosis factor αreceptor type IIwith human serum albumin.

In a further preferred embodiment, the long-acting soluble tumornecrosis factor α receptor decreases the IL-6 level in hepatocytes by40-50%;

decreases the MIP-2 level in hepatocytes by 50-60%;

decreases the bcl-xl level in hepatocytes by 30-45%; or

decreases the NF-κB level in hepatocytes by 30-45%.

In a further preferred embodiment, the hepatic failure is acute and/orsub-acute hepatic failure.

In a further preferred embodiment, the hepatocyte necrosis is massivehepatocyte necrosis.

In a further preferred embodiment, the hepatocyte necrosis is acutemassive hepatocyte necrosis.

In the second aspect, the present invention provides a pharmaceuticalcomposition comprising:

(i) an effective amount (for example, 0.00001-50wt %; more preferably,0.0001-20wt %; the most preferably, 0.001-10wt %) of a long-actingsoluble tumor necrosis factor αreceptor selected from the groupconsisting of:

a. a fusion protein of human tumor necrosis factor αreceptor type I withhuman IgG1:Fc fragment (preferably, the carboxyl terminal of the humantumor necrosis factor αreceptor type I being linked to the aminoterminal of the IgG1:Fc fragment),

b. a fusion protein of human tumor necrosis factor αreceptor type IIwith human IgG1:Fc fragment (preferably, the carboxyl terminal of thehuman tumor necrosis factor α receptor type II being linked to the aminoterminal of the IgG1:Fc fragment),

c. a human tumor necrosis factor αreceptor type I protein PEGylated atthe amino terminal,

d. a human tumor necrosis factor αreceptor type I protein PEGylated atthe carboxyl terminal,

e. a human tumor necrosis factor αreceptor type II protein PEGylated atthe amino terminal,

f. a human tumor necrosis factor αreceptor type II protein PEGylated atthe carboxyl terminal,

h. a human tumor necrosis factor αreceptor type I protein embedded in aPEG-liposome mixture,

i. a human tumor necrosis factor α receptor type II protein embedded ina PEG-liposome mixture,

j. a fusion protein of human tumor necrosis factor αreceptor type I withhuman serum albumin, or

k. a fusion protein of human tumor necrosis factor αreceptor type IIwith human serum albumin; and

(ii) a pharmaceutically acceptable vehicle.

In a further preferred embodiment, the pharmaceutical compositionfurther comprises an effective amount (for example, 0.00001-50wt %; morepreferably, 0.0001-20wt %; the most preferably, 0.001-10wt %) of one ormore agents selected from the group consisting of:

(iii) human hepatocyte growth factor (huHGF), reduced glutathione andmatrine.

In the third aspect, the present invention provides a method of treatingor preventing the hepatocyte necrosis or the hepatic failure, comprisingthe step of administering to a subject in need of such a treatment aneffective amount (for example, 0.00001-50wt %; more preferably,0.0001-20wt %; the most preferably, 0.001-10wt %) of a long-actingsoluble tumor necrosis factor αreceptor.

In a preferred embodiment, the long-acting soluble tumor necrosis factorαreceptor is selected from the group consisting of:

a. a fusion protein of human tumor necrosis factor αreceptor type I withhuman IgG1:Fc fragment (preferably, the carboxyl terminal of the humantumor necrosis factor αreceptor type 1 being linked to the aminoterminal of the IgG1:Fc fragment),

b. a fusion protein of human tumor necrosis factor αreceptor type IIwith human IgG1:Fc fragment (preferably, the carboxyl terminal of thehuman tumor necrosis factor α receptor type II being linked to the aminoterminal of the IgG1:Fc fragment),

c. a human tumor necrosis factor αreceptor type I protein PEGylated atthe amino terminal,

d. a human tumor necrosis factor αreceptor type I protein PEGylated atthe carboxyl terminal,

e. a human tumor necrosis factor αreceptor type II protein PEGylated atthe amino terminal,

f. a human tumor necrosis factor αreceptor type II protein PEGylated atthe carboxyl terminal,

h. a human tumor necrosis factor αreceptor type I protein embedded in aPEG-liposome mixture,

i. a human tumor necrosis factor α receptor type II protein embedded ina PEG-liposome mixture,

j. a fusion protein of human tumor necrosis factor αreceptor type I withhuman serum albumin, or

k. a fusion protein of human tumor necrosis factor αreceptor type IIwith human serum albumin;

In a preferred embodiment, the hepatic failure is acute and/or sub-acutehepatic failure.

MODE OF CARRYING OUT THE INVENTION

Inventors of the present invention, through a persistent and extensiveinvestigation and experiments, has found for the first time that thecontinuous and consistent block on the activity of hepatocytes inducedby the soluble TNFαreceptor is important to the treatment and/orprevention of acute massive hepatocytic necrosis. That is, it isrequired to maintain a stable and persistent level of solubleTNFαreceptor in blood and liver. In contrast, conventional TNFαreceptor,which has a short-time and pulse-moded action, is sufficient for themild hepatocyte necrosis in acute hepatitis. Thus, inventors of thepresent invention discovered the reason why the conventional soluble TNFα receptor cannot effectively prevent acute massive hepatocyte necrosisin acute hepatic failure. It is believed that its short half-life andinstability in vivo prevent it from providing a stable and sustainedaction against hepatocyte necrosis. Then, inventors of the presentinvention have modified the TNF αreceptor in different manners toprolong the acting time of TNFαreceptor in its active form in vivo andthus to produce long-acting TNFαreceptors that can provide a stable andsustained therapeutic effect. These modifications improved the effect ofTNFαreceptors in treating and preventing acute hepatocyte necrosis.Further, it is the first time that modified TNFαreceptors were used intreating acute and sub-acute hepatic failures. These constitute thebasis of the present invention.

As used herein, the term “a long-acting soluble tumor necrosis factorαreceptor” refers to a tumor necrosis factor α receptor having anprolonged half-life (that is, it can be maintained at an effectiveconcentration for a longer time in vivo). Generally, “a long-actingsoluble tumor necrosis factor αreceptor” has a half-life of more than 12hours (e.g. 12-140 hrs). Various methods can be employed to extend thehalf-life of tumor necrosis factor α receptor, including but not limitedto, fusion of the tumor necrosis factor αreceptor with the human IgG1:Fcfragment, PEGylation of the tumor necrosis factor αreceptor, embedmentof the tumor necrosis factor αreceptor within a PEG-liposome mixture andfusion of the tumor necrosis factor α receptor with the human serumalbumin. Preferably, the “long-acting soluble tumor necrosis factor αreceptor” is a “long-acting recombinant human soluble tumor necrosisfactor αreceptor”.

One object of the present invention is to provide a new pharmaceuticaluse of the gene of recombinant soluble tumor necrosis factor αreceptor(HusTNFR), particularly a new use of the gene encoding recombinantsoluble tumor necrosis factor αreceptor (HusTNFR) or the long-actingmodified recombinant human soluble tumor necrosis factor α receptor(LHusTNFR) in prevention and/or treatment of acute and sub-acute hepaticfailure.

A further object of the present invention is to provide a medicamentthat more significantly decrease the mortality of hepatic failure thanconventional soluble tumor necrosis factor αreceptors. This object ismainly achieved by increasing the half-life of the soluble tumornecrosis factor αreceptor-based agent to prolong its time of action, andthereby improving the therapeutic efficacy.

The present invention intervened the fulminant hepatic failure withlong-acting recombinant human soluble tumor necrosis factor αreceptorsin classic acute and sub-acute hepatic failure models in mouse. Theresults showed that the death rates in the intervention group and themodel group (without intervention) were 0% and 80%, respectively.

The above-said tumor necrosis factor (TNF) may be tumor necrosis factorα, which binds to a recombinant long-acting soluble protein of thecorresponding receptor on the cell membrane. The said recombinantlong-acting soluble protein of the receptor may be, for example, along-acting recombinant human soluble tumor necrosis factor αreceptortype I (LHusTNFRI) or a long-acting recombinant human soluble tumornecrosis factor αreceptor type II (LHusTNFRII), which has a half-life 10times longer than a normal recombinant human soluble tumor necrosisfactor αreceptor type I (HusTNFRI) and recombinant human soluble tumornecrosis factor α receptor type II (HusTNFRI), respectively. Theserecombinant receptors may be in the form of: (1) HusTNFRI or HusTNFRIIwith the carboxyl terminal linked to human immunoglobulin IgG:Fcfragment, or (2) HusTNFRI or

HusTNFRII with the amino terminal linked to PEG, or (3) HusTNFRI orHusTNFRII encapsuled in PEG-liposome, or (4) HusTNFRI or HusTNFRII withthe carboxyl or amino terminal linked to human serum albumin. SaidLHusTNFRI and LHusTNFRII are significantly more effective than HusTNFRIor HusTNFRII in the treatment and/or the prophylaxis of acute orsub-acute hepatic failure.

The animal model of acute or sub-acute hepatic failure may beestablished by administering D-aminogalactose and endotoxin to rats ormice) via endodermic injection. Such an animal model has a highmortality of 60-80% due to hepatic failure.

The long-acting recombinant human soluble tumor necrosis factor αreceptor (LHusTNFR) according to the present invention may be producedin the form of:

a. a recombinant protein expressed from a fusion gene of the geneencoding human TNF αreceptor (TNFR) type I and the gene encoding humanIgG1:Fc fragment,

b. a recombinant protein expressed from a fusion gene of the geneencoding human TNF αreceptor (TNFR) type II and the gene encoding humanIgG1:Fc fragment,

c. a human tumor necrosis factor αreceptor type I protein PEGylated atthe amino terminal,

d. a human tumor necrosis factor αreceptor type I protein PEGylated atthe carboxyl terminal,

e. a human tumor necrosis factor αreceptor type II protein PEGylated atthe amino terminal,

f. a human tumor necrosis factor αreceptor type II protein PEGylated atthe carboxyl terminal,

h. a human tumor necrosis factor αreceptor type I protein embedded in aPEG-liposome mixture,

i. a human tumor necrosis factor α receptor type II protein embedded ina PEG-liposome mixture,

j. a fusion protein of a human tumor necrosis factor αreceptor type Iwith human serum albumin, or

k. a fusion protein of human tumor necrosis factor αreceptor type IIwith human serum albumin.

The long-acting recombinant human soluble tumor necrosis factor αreceptor (LHusTNFR) according to the present invention, whenadministered to mice with D-aminogalactose and endotoxin-induced acutehepatic failure (herein after, referred to as “acute hepatic failureanimal(s)”) in a prophylaxis test, decreased the mortality due to acutehepatic failure from 80% to 0%. The long-acting recombinant humansoluble tumor necrosis factor α receptor (LHusTNFR) according to thepresent invention, when administered to rats with D-aminogalactose andendotoxin-induced sub-acute hepatic failure (herein after, reffered toas “sub-acute hepatic failure animal(s)”) in the prophylaxis andtreatment tests, decreased the mortality due to sub-acute hepaticfailure animal from 80% to 0%. These results show that the long-actingrecombinant human soluble tumor necrosis factor α receptors of type Iand type II, which have a longer half-life, can significantly decreasethe mortality in model animals, have excellent therapeutic effect intreatment and/or prophylaxis of acute and sub-acute hepatic failure, andare thus therapeutically and prophylactically effective against acuteand sub-acute hepatic failure. More over, such findings may lead to adevelopment of a novel medicament to significantly reduce the mortalitydue to acute hepatic failure.

According to the present invention,

(1) A gene encoding the LHusTNFR type I as set forth in SEQ ID NO: 1 isprepared. The method of preparation comprises the steps of cloning thegene encoding the extra-membrane amino acids of the sTNFR (human) type I(i.e., amino acids 1-171 of SEQ ID NO: 1) and the gene encoding the Fcfragment of the of human immunoglobulin γ1 (IgG1:Fc) (i.e., amino acidpositions 172-403 of SEQ ID NO: 1), preferably into an appropriateplasmid; identifying and screening for positive clones harboring thefusion of type I TNFR-IgG1:Fc fragment through DNA restriction enzymedigestion; and confirming the target genes via nucleotide sequencing.

(2) A gene encoding the LHusTNFR type II as set forth in SEQ ID NO: 2 isprepared. The preparation comprises the steps of cloning the geneecoding the extra-membrane amino acids of the sTNFR (human) type II(i.e. amino acids 1-235 of SEQ ID NO: 2) and the gene ecoding the Fcfragment of the of human immunoglobulin γ1 (IgG1:Fc) (i.e. amino acids236-467 of SEQ ID NO: 2) into an appropriate plasmid, identifying andscreening for the positive clones harboring the fusion of type IITNFR-IgG1:Fc fragment through DNA restriction enzyme digestion, andconfirming the target genes via nucleotid sequencing.

The obtained cDNA fragment of TNFR (I or II)-IgG1:Fe may be cloned intoan expression vector to form a recombinant expression vector such as aplasmid. According to the present invention, there is no specifictlimites on the expression plasmid. In a preferred embodiment, aprokaryotic expression vector, for example, pET28, is used.

The obtained expression vector may be introduced to an appropriate hostcell using any suitable conventional methods. The present invention isnot limited to any specific host cells. Any host, as long as it allowsthe expression of the recombinant expression vector, may be used. In apreferred embodiment, the Brewer's yeast, BL21 is used.

The expression product according to the present invention is secreted asinclusion bodies in the cytoplasm of host cells. The inclusion bodiesmay be isolated from the lysate of the host cells, and then lysed withhigh concentration of urea or guanidine hydrochloride. The LHusTNFR ispurified from the lysate of the inclusion bodies, and then renaturatedto give the active LHusTNFR (type I or II)-IgG1:Fc.

All the operations in molecular biology are carried out according to“Molecular cloning: a laboratory manual” (Sambrook and D. W. Russell,New York: Cold Spring Harbor Laboratory Press).

(3) The cDNA encoding the extra-membrane amino acids of sTNFR (human)type I (i.e., amino acids 1-171 of SEQ ID NO: 1) is cloned into aexpression vector to produce a recombinant expression plasmid (SEQ IDNO: 3)

The present invention is not limited to any specific expressionplasmids. In a preferred embodiment, a prokaryotic expression vector,for example, pET28, is used.

The said recombinant expression vector may be introduced into anappropriate host cells as previously taught. The present invention isnot limited to any specific host cells. Any host cells, as long as itallows the expression of the recombinant expression vectors can be used.In a preferred embodiment, E. coli BL21 is employed. The expressionproduct according to the present invention is secreted as inclusionbodies in the cytoplasm of the host cells. The inclusion bodies may beisolated from the lysate of the host cells, and then lysed with highconcentration of urea or guanidine hydrochloride. The LHusTNFR type I ispurified from the lysate of the inclusion bodies, and then renaturatedto give the active LHusTNFR type I.

Active mPEG(s) of molecular weight (MW) no less than 20,000 may becoupled to the amino terminal or the carboxyl terminal of HusTNFR typeI. The present invention is not limited to any specific mPEG molecules.In a preferred embodiment, mPEG2-ALD of MW 40,000 from Shearwatercorporation (New Jersey, USA) was coupled to the amino terminal ofHusTNFR type I. And, in another embodiment, an mPEG2-NHS easter of MW40,000 from Shearwater corporation (New Jersey, USA) was coupled to thecarboxyl terminal of HusTNFR type I.

The reaction can be expressed by the formula:mPEG—CHO+RNH₂+NaCNBH₃--→mPEG—CH₂—NHR

The reaction condition includes pH 7.9 and a duration of 12 hrs.

(4) The cDNA encoding the extra-membrane amino acids 1-235 of sTNFR(human) type II is cloned into an expression vector to produce arecombinant expression plasmid (SEQ ID NO: 4).

The present invention is not limited to any specific expressionplasmids. In a preferred embodiment, a prokaryotic expression vector,for example, pET28, was used.

The said recombinant expression vector may be introduced into anappropriate host cells as previously taught. The present invention isnot limited to any specific host cells. Any host cells, as long as itallows the expression of the recombinant expression vectors can be used.In a preferred embodiment, E. coli BL21 was used. The expression productaccording to the present invention is secreted as inclusion bodies intothe cytoplasm of host cells. The inclusion bodies may be isolated fromthe lysate of the host cells, and then lysed with high concentration ofurea or guanidine hydrochloride. The LHusTNFR type II is purified fromthe lysate of the inclusion bodies, and then renaturated to give theactive LHusTNFR type II.

Active mPEG(s) with a molecular weight (MW) of no less than 20,000 maybe coupled to the amino or the carboxyl terminal of HusTNFR type II. Thepresent invention is not limited to any specific mPEG molecules. In apreferred embodiment, mPEG2-ALD of MW 40,000 from Shearwater corporation(New Jersey, USA) was coupled to the amino terminal of HusTNFR type II.And in another embodiment, an mPEG2-NHS easter of MW 40,000 (Shearwatercorporation, New Jersey, USA) was coupled to the carboxyl terminal ofHusTNFR type II.

The reaction may be expressed by the formula:mPEG—CHO+RNH₂+NaCNBH₃--→mPEG—CH₂—NHR

The reaction condition includes pH 7.9 and a duration of 12 hrs.

(5) The HusTNFR type I or the HusTNFR type II is encapsulated with thelong circulating liposome-polyethylene glycol-derived phospholipid toproduce the long-acting HusTNFR type I or type II.

DOPE-PEG-MAL (WM 4108.04) is produced by reactingdioleoyl-phosphatidylethanolamine (DOPE) (WM 744.04, Aanti Polar Lipids,U.S.A.), NHS-PEG₃₅₄₀-MAL (Nhydroxysulfosuccinimide-polyoxyethylene (MW3540)-maleimide) (MW 3477, Aanti Polar Lipids, U.S.A.) and TEA in amolar ratio of 1:1:0.1 in the presence of triethylamine as the catalystat 25° C. for 6 hrs and then subjecting the resultant to centrifugation,evaporation and vacuum dehydration.

Then, the long circulating liposome-polyethylene glycol-derivedphospholipid is produced by reacting the obtained DOPE-PEG-MAL,EPC(L-α-Phosphatidylcholine, MW 760.09, cholesterol (MW 386.67), andmPEG-2000-DOPE (MW 2801.51) (Aanti Polar Lipids, U.S.A.)at a molar ratioof 200:20:10:1 in chloroform in a water bath at 40° C., and then rotaryevaporating at 100-150 r/min to remove the organic solvents, adding PBS(PH 7.4) to allow complete hydration at room temperature, extrudingrepeatedly with Mini Extruder, and passing through 100 nm filter for 15times.

Finally, the long-acting HusTNFR type I or type II is obtained by addingHusTNFR type I (SEQ ID NO:3) or HusTNFR type II (SEQ ID NO:4) in PBS tothe PBS solution of the obtained long circulating liposome-polyethyleneglycol-derived phospholipid, oscillating at 4° C. for 30 min, andpassing through CL-4B (Pharmacia, USA) to remove encapsulated HusTNFRtype I or HusTNFR type II.

(6) A gene encoding LHusTNFR type I as set forth in SEQ ID NO: 5 isprepared. The method of preparatiom comprises: cloning the encoding theextra-membrane amino acids of sTNFR (human) type I (i.e. amino acids1-171 of SEQ ID NO: 5), G(n)S-linker (i.e. amino acids 172-181) and thegene encoding human serum albumin (i.e. amino acids 182-790 of SEQ IDNO: 5) into an appropriate plasmid, identifying and screening for thepositive clones harboring the fused fragment of type I TNFR-human serumalbumin by DNA restriction enzyme digestion, and confirming the targetgene by nucleotide sequencing.

(7) A gene encoding LHusTNFR type II as set forth in SEQ ID NO: 6 isprepared. The method of preparation comprises: cloning the gene encodingthe extra-membrane amino acids of sTNFR (human) type II (i.e. aminoacids 1-235 of SEQ ID NO: 2), G(n)S-linker (i.e. amino acids 236-245)and the gene encoding human serum albumin (i.e. amino acida 246-854 ofSEQ ID NO: 6) into an appropriate plasmid, identifying and screening forthe positive clones harboring the fused fragment of type II TNFR-humanserum albumin by DNA restriction enzyme digestion, and verifying thetarget gene by nucleotide sequencing.

The fused cDNA fragments encoding the type I TNF αreceptor-human serumalbumin and type II TNF αreceptor-human serum albumin are cloned intoexpression vectors, respectively, to form recombinant expressionvectors. The present invention is not limited to any specific expressionplasmids. In a preferred embodiment, eukaryotic expression vectors usedin yeast, for example, Brewer's yeast or pichia yeast, is used.

The obtained expression vectors may be introduced into appropriate hostcells as previously taught. The present invention is not limited to anyspecific host cells. Any host cells, as long as is allow as theexpression of the recombinant expression vectors, can be used. In apreferred embodiment, the Brewer's yeast BL21 is used.

The expression product according to the present invention is secreted asinclusion bodies into the cytoplasm of the host cells. The inclusionbodies may be isolated from the lysate of the host cells, and then lysedwith high concentration of urea or guanidine hydrochloride. The LHusTNFRis purified from the lysate of the inclusion bodies, and thenrenaturated to give the active LHusTNFR.

All the operations in molecular biology are carried out according to the“Molecular cloning: A laboratory manual” (Sambrook and D. W. Russell,New York: Cold Spring Harbor Laboratory Press).

As can be measured, the HusTNFR-IgG type I: and type II:Fc fusionproteins are maintained at effective levels in vivo for 70 to 90 hrs(i.e., a half-life of 35 to 40 hrs). The fusion protein of HusTNFR typeII-human serum albumin can be maintained at effective levels in vivo forabout 60 hrs (i.e., a half-life of about 30 hrs). The PEG-HusTNFR andthe HusTNFR encapsuled within the long circulating liposome-polyethyleneglycol-derived phospholipid can be maintained at effective levels invivo for 6 to 11 days (i.e., a half-life of 3 to 5.5 days).

It can be seen that all the LHusTNFRs produced in different formsaccording to methods described above each have a half-life of more than12 hrs (12 to 140 hrs), and hence meet the criterion of long-action. Incontrast, the normal soluble tumor necrosis factor a receptor has ahalf-life of only 50 minutes to 2 hours.

LHusTNFR prepared by the genetic engineering method according to thepresent invention can efficiently prevent and treat the acute and thesub-acute hepatic failure. Comparison of properties with theconventional HusTNFR shows that the LHusTNFR has a significantlyprolonged half-life and a notable improvement in the effect ofpreventing and/or treating the acute and the sub-acute hepatic failureand decreasing associated mortality.

The present invention further provides a pharmaceutical composition fortreating the hepatocyte necrosis or the hepatic failure, which comprisesthe long-acting soluble tumor necrosis factor α receptor described aboveand a pharmaceutically acceptable vehicle. Generally, the composition isformulated in an atoxic, inert and pharmaceutically acceptable aqueousmedium, wherein the pH value may be adjusted depending on the nature ofthe components in the formulation and/or the condition to be treated,and is typically about 5-8, preferably about 6-8. The formulatedpharmaceutical composition can be administered in any suitable ways,including but not limited to intraperitoneal, intravenous or topicalroutes.

The composition according to the present invention may be directly usedto treat hepatocyte necrosis or hepatic failure. Also, the compositionmay be administered in combination with other therapeutic agents such asthe human hepatocyte growth factor (huHGF), the reduced glutathione andmatrine, etc.

The pharmaceutical composition according to the present inventioncomprises a safe and effective amount of the long-acting soluble tumornecrosis factor αreceptor according to the present invention and apharmaceutically acceptable vehicle or excipient. The said vehicle maybe but not limited to a saline solution, a buffer, glucose, water,glycerin, ethanol, and a mixture thereof. The pharmaceutical compositionmay be formulated into appropriate forms suitable for the intendedadministration routes. The pharmaceutical composition according to thepresent invention may be in thea form suitable for injection, which maybe prepared in saline or an aqueous solution comprising glucose andother excipients. The pharmaceutical composition, such as an injectableformulation or a solution, is preferably prepared under a sterilecondition. The amount of active ingredients to be administered dependson the therapeutically effective amount. For example, the amount ofadministration may be about 0.1 microgram/kilogram body weight/day to 5milligram/kilogram body weight/day.

When the pharmaceutical composition is used, a safe and effective amountof the long-acting soluble tumor necrosis factor αreceptor according tothe present invention is administered to a mammal. Typically, the saidsafe and effective amount is at least about 1 microgram/kilogram bodyweight, while not exceeding 8 microgram/kilogram body weight in mostcases. Preferably, the said safe and effective amount is about 10microgram/kilogram body weight to about 1 milligram/kilogram bodyweight. Obviously, the specific dosage can readily be determined by aphysician according to the factors such as the administration route andthe physical condition of the patient.

The present invention will be further illustrated with the followingexamples. It should be understood that, these examples are exemplaryonly and are not intended to limit the scope of the present invention.The experimental methods in the following examples not indicating thespecific experimental conditions are typically carried out under theconventional conditions, for example, those in Sambrook, et al.Molecular cloning: A laboratory manual (New York: Cold Spring HarborLaboratory Press, 1989), or following the manufacture's instructions.

EXAMPLE 1 Prevention of Acute (Fulminant) Hepatic Failure in Mice UsingLong-acting Recombinant Human Soluble Tumor Necrosis Factor (TNF) αReceptor (LHusTNFR) Type I in the Form of HusTNFR Type I-IgG1:Fc

Fulminant hepatic failure was induced via subcutaneous injection ofeither D-aminogalactose/endotoxin (GaIN/LPS) or Con-A (T cell mitogensconcanavalin A) in mice. The mortality measure after 48 hrs was 80% and50%, respectively. The gross specimen of liver exhibited severecongestion and intumesce. The HE-staining of the pathological sectionshowed massive severe hepatocyte necrosis.

In the group of prevention using the long-acting receptor of type I,C57BL/6 mice (GaIN/LPS sub-group) and BALB/c mice (Con-A sub-group) weresubcutaneously injected with 12.5 mg/kg of the LHusTNFR type I preparedas described above (i.e., the fusion protein of LHusTNFR-IgG1 type I:Fcprepared according to the method in paragraph (1) above, used as aprophylactic agent). The C57BL/6 mice (GaIN/LPS sub-group)and the BALB/cmice (Con-A sub-group) in the group of prevention using the conventionalreceptor of type I were subcutaneously injected with 12.5 mg/kg of theconventional HusTNFR. The same animals in the control group weresubcutaneously injected with saline at the same volume. 16 hrs later,the prevention groups and the control group were subcutaneously injectedwith GaIN/LPS or Con-A. The mortality after 48 hrs was observed to be80% in the GaIN/LPS sub-group in the control group (50% in the Con-Asub-group), 50% in the prevention group using the conventional receptortype I (30% for Con-A sub-group), 0% for prevention group using thelong-acting receptor of type I (0% for Con-A sub-group). In theprevention group using the long-acting receptor of type I, the pathologyexamination showed mild liver congestion and intumesce, the HE stainingshowed mild and spotty necrosis, and the massive hepatocyte necrosis asseen in the control group was not observed. The IL-6, MIP-2 and bcl-xLmRNA levels were measured by real-time PCR using the total RNAs and thenucleic proteins extracted from the liver. NF-kB level was measured byEMSA. The results showed that IL-6, MIP-2 and bcl-xL mRNA levels in theprevention group using the long-acting receptor type I (GaIN/LPSsub-group) were decreased by 82.3%, 78.1% and 84.3% as compared with thecontrol goup, and 44%, 52.2% and 37.8% as compared with the preventiongroup using the conventional receptor of type I. And, the NF-kB levelwas decreased by 87.4% and 37.5% respectively in the comparison. Theabove results indicate that the soluble TNFαreceptor type I can blockthe signaling to the nucleus mediated by TNFα binding to the type Ireceptor on the hepatic cell membrane by inhibiting the binding ofTNFαto the membrane receptor, and thus prevent the hepatocytic necrosisand the necrosis-associated acute hepatic failure. The results alsoindicate that the long-acting soluble TNFα receptor type I has asignificantly improved effect in the prevention of acute hepatic failureas compared with the conventional soluble TNFα receptor type I.

EXAMPLE 2 Prevention of Acute (Fulminant) Hepatic Failure in Mice UsingLong-acting Recombinant Human Soluble Tumor Necrosis Factor (TNF) αReceptor (LHusTNFR) Type II in the Form of HusTNFR Type II -IgG1:Fc

Fulminant hepatic failure was induced via subcutaneous injection ofeither D-aminogalactose/endotoxin (GaIN/LPS) or Con-A (T cell mitogensconcanavalin A) in mice. The mortality measure after 48 hrs was 80% and50%, respectively. The gross specimen of liver exhibited severecongestion and intumesce. The HE-staining of the pathological sectionshowed massive severe hepatocyte necrosis.

In the group of prevention using the long-acting receptor of type II,C57BL/6 mice (GaIN/LPS sub-group) and BALB/c mice (Con-A sub-group) weresubcutaneously injected with 12.5 mg/kg of the LHusTNFR type II preparedas described above (i.e., the fusion protein of LHusTNFR type II-IgG1:Fcprepared according to the method in paragraph (2) above, used as aprophylactic agent). The C57BL/6 mice (GaIN/LPS sub-group) and theBALB/c mice (Con-A sub-group) in the group of prevention using theconventional receptor of type II were subcutaneously injected with 12.5mg/kg of the conventional HusTNFR. The same animals in the control groupwere subcutaneously injected with saline at the same volume. 16 hrslater, the prevention groups and the control group were subcutaneouslyinjected with GaIN/LPS or Con-A. The mortality after 48 hrs was observedto be 80% in the GaIN/LPS sub-group in the control group (50% in theCon-A sub-group), 50% in the prevention group using the conventionalreceptor type II (30% for Con-A sub-group), 0% for prevention groupusing the long-acting receptor of type II (0% for Con-A sub-group). Inthe prevention group using the long-acting receptor of type II, thepathology examination showed mild liver congestion and intumesce, the HEstaining showed mild and spotty necrosis, and the massive hepatocytenecrosis as seen in the control group was not observed. The IL-6, MIP-2and bcl-xL mRNA levels were measured by real-time PCR using the totalRNAs and the nucleic proteins extracted from the liver. NF-kB level wasmeasured by EMSA. The results showed that IL-6, MIP-2 and bcl-xL mRNAlevels in the prevention group using the long-acting receptor type II(GaIN/LPS sub-group) were decreased by 78,3%, 72.1%, and 77.3%, ascompared with the control group, and 44%, 52.2% and 37.8% as comparedwith the prevention group using the conventional receptor type II. And,the NF-kB level was decreased by 78.4% and 37.5% respectively in thecomparison. The above results indicate that the soluble TNFαreceptortype II can block the signaling to the nucleus mediated by TNFα bindingto the type II receptor on the hepatic cell membrane by inhibiting thebinding of TNFαto the membrane receptor, and thus prevent thehepatocytic necrosis and the necrosis-associated acute hepatic failure.The results also indicate that the long-acting soluble TNFα receptortype II has a significantly improved effect in the prevention of acutehepatic failure as compared with the conventional soluble TNFα receptortype II.

EXAMPLE 3 Prevention of Acute (Fulminant) Hepatic Failure in Mice UsingLong-acting Recombinant Human Soluble Tumor Necrosis Factor (TNF) αReceptor (LHusTNFR) Type II in the Form of HusTNFR Type II-Human SerumAlbumin

Fulminant hepatic failure was induced via subcutaneous injection ofeither D-aminogalactose/endotoxin (GaIN/LPS) or Con-A (T cell mitogensconcanavalin A) in mice. The mortality measure after 48 hrs was 80% and50%, respectively. The gross specimen of liver exhibited severecongestion and intumesce. HE-staining of the pathological section showedmassive severe hepatocyte necrosis.

In the group of prevention using the long-acting receptor of type II,C57BL/6 mice (GaIN/LPS sub-group) and BALB/c mice (Con-A sub-group) weresubcutaneously injected with 5-30 mg/kg of the LHusTNFR type II preparedas described above (i.e., the fusion protein of LHusTNFR type II-humanserum albumin prepared according to the method in paragraph (6) above,used as a prophylactic agent). The C57BL/6 mice (GaIN/LPS sub-group)andthe BALB/c mice (Con-A sub-group) in the group of prevention using theconventional receptor of type II were subcutaneously injected with 12.5mg/kg of the conventional HusTNFR. The same animals in the control groupwere subcutaneously injected with saline at the same volume, 16 hrslater, the prevention groups and the control group were subcutaneouslyinjected with GaIN/LPS or Con-A. The mortality after 48 hrs was observedto be 80% in the GaIN/LPS sub-group in the control group (50% in theCon-A sub-group), 50% in the prevention group using the conventionalreceptor type II (30% for Con-A sub-group), 0% for prevention groupusing the long-acting receptor of type II (0% for Con-A sub-group). Inthe prevention group using the long-acting receptor of type II, thepathology examination showed mild liver congestion and intumesce, the HEstaining showed mild and spotty necrosis, and the massive hepatocytenecrosis as seen in the control group was not observed. The IL-6, MIP-2and bcl-xL mRNA levels were measured by real-time PCR using the totalRNAs and the nucleic proteins extracted from the liver. NF-kB level wasmeasured by EMSA. The results showed that IL-6, MIP-2 and bcl-xL mRNAlevels in the prevention group using the long-acting receptor type II(GaIN/LPS sub-group) were significantly decreased by 30-60%, as comparedwith the prevention group using the conventional receptor type II andthe control group. And, the NF-kB level was decreased by 60% and 32%respectively in the comparison. The above results indicate that the TNFRtype II-human serum albumin can block the signaling to the nucleusmediated by TNFα binding to the type II receptor on the hepatic cellmembrane by inhibiting the binding of TNFαto the membrane receptor, andthus prevent the hepatocytic necrosis and the necrosis-associated acutehepatic failure. The results also indicate that the long-acting solubleTNFαreceptor type II has a significantly improved effect in theprevention of acute hepatic failure as compared with the conventionalsoluble TNFαreceptor type II.

EXAMPLE 4 Prevention of Sub-acute Hepatic Failure in Rats UsingLong-acting Recombinant Human Soluble Tumor Necrosis Factor (TNF)αReceptor (LHusTNFR) Type I in the Form of HusTNFR Type I-IgG1:Fc

Sub-acute hepatic failure was induced via subcutaneous injection withD-aminogalactose/endotoxin (GaIN/LPS) in the rats. The mortality afterone week was 60%. The gross specimen of liver exhibited severecongestion and intumesce. The HE-staining of the pathological sectionshowed a massive severe hepatocyte necrosis.

In the prevention group using the long-acting receptor type I, theSpraque-Dawley rats were subcutaneously injected with 12.5 mg/kg of thetype I LHusTNFR prepared according to the present invention (i.e., thefusion protein of LHusTNFR type I-IgG1:Fc prepared according to themethod in paragraph (1) above, used as a prophylactic agent). TheSpraque-Dawley rats in the group of prevention using the conventionalreceptor type I were subcutaneously injected with 12.5 mg/kg of theconventional HusTNFR. The same animals in the control group weresubcutaneously injected with saline at the same volume. 16 hrs later,the prevention groups and the control group were subcutaneously injectedwith GaIN/LPS. After one week, the mortality was observed to be 60% inthe control group, 44% in the prevention group using the conventionalreceptor type I, and 0% in the prevention group using the long-actingreceptor type I. In the prevention group using the long-acting receptortype I, the pathology examinatin showed mild liver congestion andintumesce, the HE staining showed mild and spotty necrosis, and themassive hepatocyte necrosis as seen in the control group was notobserved. The IL-6, MIP-2 and bcl-xL mRNA levels were measured byreal-time PCR using the total RNAs and the nucleic proteins extractedfrom the liver. NF-kB level was measured by EMSA. The results showedthat IL-6, MIP-2 and bcl-xL mRNA levels in the prevention group usingthe long-acting receptor type I were decreased by 90.5%, 78.1% and 84.3%as compared with the prevention group using the conventional receptortype I, and 46.7%, 52.2% and 37.8% as compared with the control group.And, the NF-kB level was decreased by 87.4% and 37.5% respectively inthe comparison. The above results indicate that the soluble TNFαreceptortype I can block the signaling to nucleus mediated by the TNFαbinding tothe receptor of type I on the hepatic cell membrane by inhibiting thebinding of TNFαto the membrane receptor, and thus prevent thehepatocytic necrosis and the necrosis-associated acute hepatic failure.The results also indicate that the long-acting soluble TNFα receptortype I has a significantly improved effect in the prevention ofsub-acute hepatic failure as compared with the conventional soluble TNFαreceptor type I.

EXAMPLE 5 Prevention of Sub-acute Hepatic Failure in Rats UsingLong-acting Recombinant Human Soluble Tumor Necrosis Factor (TNF)αReceptor (LHusTNFR) Type II in the Form of HusTNFR Type II -IgG1:Fc

Sub-acute hepatic failure was induced via subcutaneous injection withD-aminogalactose/endotoxin (GaIN/LPS) in the rats. The mortality afterone week was 60%. The gross specimen of liver exhibited severecongestion and intumesce. The HE-staining of the pathological sectionshowed a massive severe hepatocyte necrosis.

In the prevention group using the long-acting receptor type II, theSpraque-Dawley rats were subcutaneously injected with 12.5 mg/kg of thetype II LHusTNFR prepared according to the present invention (i.e., thefusion protein of LHusTNFR type II -IgG1:Fc prepared according to themethod in paragraph (2) above, used as a prophylactic agent). TheSpraque-Dawley rats in the group of prevention using the conventionalreceptor type II were subcutaneously injected with 12.5 mg/kg of theconventional HusTNFR. The same animals in the control group weresubcutaneously injected with saline at the same volume. 16 hrs later,the prevention groups and the control group were subcutaneously injectedwith GaIN/LPS. After one week, the mortality was observed to be 60% inthe control group, 44% in the prevention group using the conventionalreceptor type II, and 0% in the prevention group using the long-actingreceptor type II. In the prevention group using the long-acting receptortype II, the pathology examinatin showed mild liver congestion andintumesce, the HE staining showed mild and spotty necrosis, and themassive hepatocyte necrosis as seen in the control group was notobserved. The IL-6, MIP-2 and bcl-xL mRNA levels were measured byreal-time PCR using the total RNAs and the nucleic proteins extractedfrom the liver. NF-kB level was measured by EMSA. The results showedthat IL-6, MIP-2 and bcl-xL mRNA levels in the prevention group usingthe long-acting receptor type II were decreased by 88.5%, 68.1% and78.3% as compared with the prevention group using the conventionalreceptor type II, and 46.7%, 52.2% and 37.8% as compared with thecontrol group. And, the NF-kB level was decreased by 92.1% and 37.5%respectively in the comparison. The above results indicate that thesoluble TNFαreceptor type II can block the signaling to nucleus mediatedby the TNFαbinding to the receptor of type II on the hepatic cellmembrane by inhibiting the binding of TNFαto the membrane receptor, andthus prevent the hepatocytic necrosis and the necrosis-associatedsub-acute hepatic failure. The results also indicate that thelong-acting soluble TNFαreceptor type II has a significantly improvedeffect in the prevention of sub-acute hepatic failure as compared withthe conventional soluble TNFα receptor type II.

EXAMPLE 6 Prevention of Sub-acute Hepatic Failure in Rats UsingLong-acting Recombinant Human Soluble Tumor Necrosis Factor (TNF)αReceptor (LHusTNFR) Type II in the Form of HusTNFR type II-Human SerumAlbumin

Sub-acute hepatic failure was induced via subcutaneous injection withD-aminogalactose/endotoxin (GaIN/LPS) in the rats. The mortality afterone week was 60%. The gross specimen of liver exhibited severecongestion and intumesce. The HE-staining of the pathological sectionshowed a massive severe hepatocyte necrosis.

In the prevention group using the long-acting receptor type II, theSpraque-Dawley rats were subcutaneously injected with 5-30 mg/kg of thetype II LHusTNFR prepared according to the present invention (i.e., thefusion protein of LHusTNFR type II-human serum albumin preparedaccording to the method in paragraph (6) above, used as a prophylacticagent). The Spraque-Dawley rats in the group of prevention using theconventional receptor type II were subcutaneously injected with 12.5mg/kg of the conventional HusTNFR. The same animals in the control groupwere subcutaneously injected with saline at the same volume. 16 hrslater, the prevention groups and the control group were subcutaneouslyinjected with GaIN/LPS. After one week, the mortality was observed to be60% in the control group, 44% in the prevention group using theconventional receptor type II, and 0% in the prevention group using thelong-acting receptor type II. In the prevention group using thelong-acting receptor type II, the pathology examinatin showed mild livercongestion and intumesce, the HE staining showed mild and spottynecrosis, and the massive hepatocyte necrosis as seen in the controlgroup was not observed. The IL-6, MIP-2 and bcl-xL mRNA levels weremeasured by real-time PCR using the total RNAs and the nucleic proteinsextracted from the liver. NF-kB level was measured by EMSA. The resultsshowed that IL-6, MIP-2 and bcl-xL mRNA levels in the prevention groupusing the long-acting receptor type II were significantly decreased by25-55% as compared with the prevention group using the conventionalreceptor type II and the control group. And, the NF-kB level wasdecreased by 51% and 27% respectively in the comparison. The aboveresults indicate that the soluble TNFαreceptor type II can block thesignaling to nucleus mediated by the TNFαbinding to the receptor of typeII on the hepatic cell membrane by inhibiting the binding of TNFαto themembrane receptor, and thus prevent the hepatocytic necrosis and thenecrosis-associated sub-acute hepatic failure. The results also indicatethat the long-acting soluble TNFα receptor type II has a significantlyimproved effect in the prevention of sub-acute hepatic failure ascompared with the conventional soluble TNFαreceptor type II.

EXAMPLE 7 Treatment of Sub-acute Hepatic Failure in Rats UsingLong-acting Recombinant Human Soluble Tumor Necrosis Factor (TNF)Preceptor (LHusTNFR) Type I in the Form of HusTNFR Type I-IgG1:Fc

Sub-acute hepatic failure was induced via subcutaneous injection withD-aminogalactose/endotoxin (GaIN/LPS). The mortality after one week was60%. The specimen of liver exhibited severe congestion and intumesce.The HE-staining of the pathological section showed a massive severehepatocyte necrosis.

The rat model of sub-acute hepatic failure was made by subcutaneousinjection with GaIN/LPS. After 8 hrs, in the group treated withlong-acting receptor of type I, the Spraque-Dawley rates weresubcutaneously injected with 12.5 mg/kg of LHusTNFR type I preparedaccording to the present invention (i.e., the fusion protein of LHusTNFRtype I-IgG1:Fc produced according to the method in paragraph (1)). TheSpraque-Dawley rats in the group treated with the conventional receptorof type I were subcutaneously injected with 12.5 mg/kg of theconventional HusTNFR. The same animals in the control group weresubcutaneously injected with saline at the same volume. The rats wereobserved for one week. Within one week post GaIN/LPS injection, themortality was observed to be 60% in the control group, 30% in the grouptreated with the conventional receptor type I, 0% in the group treatedwith the long-acting receptor type I. In the group of treated with thelong-acting receptor type I, the pathology examination showed only mildliver congestion and intumesce, the HE staining showed mild and spottynecrosis, and the massive hepatocyte necrosis as seen in the controlgroup was not observed. The IL-6, MIP-2 and bcl-xL mRNA levels weremeasured by real-time PCR using the total RNAs and the nucleic proteinsextracted from the liver. The NF-kB level was measured by EMSA. Theresults showed that IL-6, MIP-2 and bcl-xL mRNA levels in the grouptreated with the long-acting receptor type I were decreased by 90.5%,78.1% and 84.3% as compared with the group treated with the conventionalreceptor type I, and 46.7%, 52.2% and 37.8% as compared with the controlgroup. And, the NF-kB level was decreased by 87.4% and 37.5%respectively in the compasition. The results above indicate that thesoluble TNFαreceptor type I can block the signaling to the nucleusmediated by TNFα binding to the type I receptor on the hepatic cellmembrane by inhibiting the binding of TNFαto the membrane receptor, andthus prevent the hepatocytic necrosis and the necrosis-associatedsub-acute hepatic failure. The results also indicates that thelong-acting soluble TNFαreceptor type I has a significantly improvedeffect in the treatment of sub-acute hepatic failure as compared withthe conventional soluble TNFαreceptor type I.

EXAMPLE 8 Treatment of Sub-acute Hepatic Failure In Rats UsingLong-acting Recombinant Human Soluble Tumor Necrosis Factor (TNF)αReceptor (LHusTNFR) Type II in the Form of HusTNFR Type II-IgG1:Fc

Sub-acute hepatic failure was induced via subcutaneous injection withD-aminogalactose/endotoxin (GaIN/LPS). The mortality after one week was60%. The specimen of liver exhibited severe congestion and intumesce.The HE-staining of the pathological section showed a massive severehepatocyte necrosis.

The rat model of sub-acute hepatic failure was made by subcutaneousinjection with GaIN/LPS. After 8 hrs, in the group treated withlong-acting receptor of type II, the Spraque-Dawley rates weresubcutaneously injected with 12.5 mg/kg of LHusTNFR type II preparedaccording to the present invention (i.e., the fusion protein of LHusTNFRtype II-IgG1:Fc produced according to the method in paragraph (2)). TheSpraque-Dawley rats in the group treated with the conventional receptorof type II were subcutaneously injected with 12.5 mg/kg of theconventional HusTNFR. The same animals in the control group weresubcutaneously injected with saline at the same volume. The rats wereobserved for one week. Within one week post GaIN/LPS injection, themortality was observed to be 60% in the control group, 30% in the grouptreated with the conventional receptor type II, 0% in the group treatedwith the long-acting receptor type II. In the group of treated with thelong-acting receptor type II, the pathology examination showed only mildliver congestion and intumesce, the HE staining showed mild and spottynecrosis, and the massive hepatocyte necrosis as seen in the controlgroup was not observed. The IL-6, MIP-2 and bcl-xL mRNA levels weremeasured by real-time PCR using the total RNAs and the nucleic proteinsextracted from the liver. The NF-kB level was measured by EMSA. Theresults showed that IL-6, MIP-2 and bcl-xL mRNA levels in the grouptreated with the long-acting receptor type II were decreased by 89%, 76%and 83% as compared with the group treated with the conventionalreceptor type II, and 44.3%, 49% and 35.2% as compared with the controlgroup. And, the NF-kB level was decreased by 79.6% and 36% respectivelyin the compasition. The results above indicate that the solubleTNFαreceptor type II can block the signaling to the nucleus mediated byTNFα binding to the type II receptor on the hepatic cell membrane byinhibiting the binding of TNFαto the membrane receptor, and thus preventthe hepatocytic necrosis and the necrosis-associated sub-acute hepaticfailure. The results also indicates that the long-acting solubleTNFαreceptor type II has a significantly improved effect in thetreatment of sub-acute hepatic failure as compared with the conventionalsoluble TNFαreceptor type II.

EXAMPLE 9 Treatment of Sub-acute Hepatic Failure in Rats UsingLong-acting Recombinant Human Soluble Tumor Necrosis Factor (TNF)αReceptor (LHusTNFR) Type II in the Form of HusTNFR Type II-Human SerumAlbumin

Sub-acute hepatic failure was induced via subcutaneous injection withD-aminogalactose/endotoxin (GaIN/LPS). The mortality after one week was60%. The specimen of liver exhibited severe congestion and intumesce.The HE-staining of the pathological section showed a massive severehepatocyte necrosis.

The rat model of sub-acute hepatic failure was made by subcutaneousinjection with GaIN/LPS. After 8 hrs, in the group treated withlong-acting receptor of type II, the Spraque-Dawley rates weresubcutaneously injected with 5-30 mg/kg of LHusTNFR type II preparedaccording to the present invention (i.e., the fusion protein of LHusTNFRtype II-human serum albumin produced according to the method inparagraph (6)). The Spraque-Dawley rats in the group treated with theconventional receptor of type II were subcutaneously injected with 12.5mg/kg of the conventional HusTNFR. The same animals in the control groupwere subcutaneously injected with saline at the same volume. The ratswere observed for one week. Within one week post GaIN/LPS injection, themortality was observed to be 60% in the control group, 30% in the grouptreated with the conventional receptor type II, 0% in the group treatedwith the long-acting receptor type II. In the group of treated with thelong-acting receptor type II, the pathology examination showed only mildliver congestion and intumesce, the HE staining showed mild and spottynecrosis, and the massive hepatocyte necrosis as seen in the controlgroup was not observed. The IL-6, MIP-2 and bcl-xL mRNA levels weremeasured by real-time PCR using the total RNAs and the nucleic proteinsextracted from the liver. The NF-kB level was measured by EMSA. Theresults showed that IL-6, MIP-2 and bcl-xL mRNA levels in the grouptreated with the long-acting receptor type II were significantlydecreased by 20-50% as compared with the group treated with theconventional receptor type II and the control group. And, the NF-kBlevel was decreased by 41% and 24% respectively in the compasition. Theresults above indicate that the soluble TNFαreceptor type II can blockthe signaling to the nucleus mediated by TNFα binding to the type IIreceptor on the hepatic cell membrane by inhibiting the binding ofTNFαto the membrane receptor, and thus prevent the hepatocytic necrosisand the necrosis-associated sub-acute hepatic failure. The results alsoindicates that the long-acting soluble TNFαreceptor type II has asignificantly improved effect in the treatment of sub-acute hepaticfailure as compared with the conventional soluble TNFα receptor type II.

All references mentioned in this application are herein incorporated byreference into the specification to the same extent as if each wasspecifically and individually indicated to be incorporated herein byreference. Additionally, it will be understood that in light of theabove disclosure of the present invention, those skilled in the art canmake various changes and modifications, all of which are falling in thescope of the claims of the present invention.

The invention claimed is:
 1. A method of preventing acute or sub-acutehepatic failure in a subject that does not have acute or sub-acutehepatic failure, comprising: administering to the subject that does nothave acute or sub-acute hepatic failure a fusion protein of human tumornecrosis factor α receptor II with human IgG1:Fc fragment in an amounteffective for preventing acute or sub-acute hepatic failure.
 2. Themethod of claim 1, wherein the fusion protein has a sequence of SEQ IDNO:
 2. 3. The method of claim 1, wherein administering to the subjectcomprises administering the amount of the fusion protein beforeadministering a drug that is capable of inducing hepatic failure in thesubject.
 4. The method of claim 3, wherein the drug is at least oneselected from the group consisting of D-aminogalactose, endotoxin and Tcell mitrogens concanavalin A.
 5. The method of claim 1, wherein theamount of the fusion protein is an amount effective for suppressing theexpression of at least one protein selected from the group consisting ofIL-6, TNF-α, MIP-2, bcl-xL and NF-κB.